Method for finger-printing heparins

a fingerprinting and heparin technology, applied in the field of fingerprinting heparins, can solve the problems of increased bleeding risk, immunological reaction, patient-to-patient response variability, etc., and achieve the effect of accurate and consistent fingerprinting, high level of resolution, and polydisperse heparin mixtures

Inactive Publication Date: 2011-03-03
VIRGINIA COMMONWEALTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]A method has been developed to accurately and consistently fingerprint unfractionated, polydisperse heparin mixtures. The method resolves broad heterogeneous heparin peaks into numerous (>20) components in a reliable, highly reproducible manner. This high level of resolution is attained by adding to the heparin sample polyamine resolving agents. When the heparin/polyamine mixture is analyzed using, for example, capillary electrophoresis (CE), the resulting electropherogram constitutes a high-resolution “fingerprint” that is characteristic of the heparin sample. Comparison of the fingerprints from two different heparin samples can reveal whether the two samples are the same

Problems solved by technology

Yet, they suffer from significant problems including enhanced bleeding risk, immunological reaction, patient-to-patient response variability, narrow therapeutic index, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio.
The many adverse side effects of heparin and LMWH arise from their structure.
Yet, these interactions are different and unpredictable for different heparin or LMWH chains because their microscopic structures are different.
These differences are perhaps the single major source of complications associated with heparin therapy.
Thus, patients on enoxaparin may not be routinely switched to tinzaparin and vice versa.
Both these techniques resolve UFH and LMWH into oligomers, especially the smaller chains, but do not provide more detailed str

Method used

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  • Method for finger-printing heparins
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  • Method for finger-printing heparins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Fingerprinting of Lovenox®

[0059]Three solutions were prepared. Solution A was 13.5 mg Lovenox® previously dialyzed to eliminate packaging excipients was dissolved in 260 μL of water; solution B was 25 mg of sodium cyanoborohydride in 300 μl of water; and solution C was 4 mg AMAC (FIG. 7) dissolved in 158 μL of 85% (v / v) acetic acid:DMSO. Solutions A, B and C were mixed and allowed to incubate at 37° C. for 16 hours. Following incubation, the mixture was dialyzed against high purity water to remove free, unreacted AMAC, followed by lyophilization. The solid powder so obtained was dissolved in high purity, deionized water containing 10% DMSO (v / v) at 10 mg / mL and stored at −78° C. until use.

[0060]CE was performed using a 75 μm fused silica capillary (40 cm effective length to the detector window) installed in a Beckman-Coulter P / ACE MDQ capillary electrophoresis system. A fresh capillary was activated using a 5 min flush of 1M NaOH, deionized water, 1M H3PO4, and deionized water each ...

example 2

Different LMWHs Display Different Fingerprint Patterns

[0061]To assess whether fingerprint pattern is characteristic of individual LMWHs, we compared CE runs of enoxaparin, tinzaparin, and Sigma LMWH in the presence of 50 IM 4EP at pH 2.3 (FIG. 9). As can be seen, each LMWH shows a characteristic fingerprint pattern defined primarily by the extent of interaction with the resolving agent. Whereas enoxaparin displays prominent peaks at 25 and 30 min, Sigma LMWH is devoid of the pattern at 30 min. In contrast, both these patterns are absent in tinzaparin. Also, tinzaparin displays much lower resolution than enoxaparin and Sigma LMWH. Equivalent results were observed for other resolving agents including SPM and 5EH (not shown).

example 3

Consistency of Results Over Time

[0062]In this experiment, a generic form of LMWH from Sigma (labeled as Sigma LMWH) was used. As in Example 1, three solutions were prepared. Solution A was 35 mg Sigma LMWH (ID# H-3400), without pre-dialysis, dissolved in 500 μL of water; solution B was 63 mg of sodium cyanoborohydride in 750 μL of water; and solution C was 10 mg AMAC in 400 μL of 85% (v / v) acetic acid:DMSO. Solutions A, B, and C were mixed and allowed to incubate at 37° C. for 16 hours. Following incubation, the mixture was dialyzed and lyophilized in a manner identical to that described in Example 1. The solid powder so obtained was dissolved in high purity, deionized water to form a 10 mg / ml stock solution and stored −78° C. until use.

[0063]Three consecutive runs were performed to assess reproducibility of the electrophoretic profile. The fingerprints are shown in FIG. 10. As can be seen, the fingerprints were highly reproducible from run to run with an intraday variation of less ...

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Abstract

Methods to generate a distinctive fingerprint (pattern of migration) for a sample of complex, polydisperse heparins are provided. The methods involve adding resolving agents such as polyamines to a heparin sample and then analyzing the sample with a technique that separates macromolecules according to charge to mass ratio (e.g. capillary electrophoresis). The resulting electropherogram is unique to and characteristic of the heparin sample. The methods may be used, for example, to monitor the quality and consistency of various heparin preparations.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The invention generally relates to methods to “fingerprint” or resolve the migration patterns of complex, polydisperse heparin mixtures. In particular, the invention provides methods for resolving complex heparins by adding polyamine resolving agents to the mixture prior to analysis of the heparins by a technique that separates macromolecules according to charge to mass ratio.[0003]2. Background of the Invention[0004]Anticoagulants are molecules used to treat and prevent a number of thrombotic disorders including pulmonary embolism, deep-vein thrombosis, disseminated intravascular coagulation, acute myocardial infarction, unstable angina, cerebrovascular thrombosis, and others. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US (1). Thus, a huge market exists for anticoagulants.[0005]The two most commonly used anticoag...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N27/447G01N33/5308G01N2400/40C08B37/0075C08K5/17C08L79/02C08G73/0206C08L5/10C08K5/3467
Inventor DESAI, UMESH R.KING, J. TIMOTHY
Owner VIRGINIA COMMONWEALTH UNIV
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